Conjugative transfer of ICESde3396 between three β-hemolytic streptococcal species

Abstract

Background: Integrative conjugative elements (ICEs) are mobile genetic elements (MGEs) that possess all genes necessary for excision, transfer and integration into recipient genome. They also carry accessory genes that impart new phenotypic features to recipient strains. ICEs therefore play an important role in genomic plasticity and population structure. We previously characterised ICESde3396, the first ICE identified in the β-hemolytic Streptococcus dysgalactiae subsp equisimilis (SDSE) and demonstrated its transfer to single isolates of Streptococcus pyogenes (group A streptococcus, GAS) and Streptococcus agalactiae (group B streptococcus, GBS). While molecular studies found the ICE in multiple SDSE and GBS isolates, it was absent in all GAS isolates examined.

Results: Here we demonstrate that ICESde3396:km is transferable from SDSE to multiple SDSE, GAS and GBS isolates. However not all strains of these species were successful recipients under the same growth conditions. To address the role that host factors may have in conjugation we also undertook conjugation experiments in the presence of A549 epithelial cells and DMEM. While Horizontal Gene Transfer (HGT) occurred, conjugation efficiencies were no greater than when similar experiments were conducted in DMEM. Additionally transfer to GAS NS235 was successful in the presence of DMEM but not in Todd Hewitt Broth suggesting that nutritional factors may also influence HGT. The GAS and GBS transconjugants produced in this study are also able to act as donors of the ICE.

Conclusion: We conclude that ICEs are major sources of interspecies HGT between β-hemolytic streptococci, and by introducing accessory genes imparting novel phenotypic characteristics, have the potential to alter the population structure of these species.

Figure 1

Schematic diagram of ICE Sde 3396:km (not to scale). The three regions (Region 1, Region 2 and Region 3) of the ICE, and regions targeted by PCR to determine the presence of these regions (i.e. R1, R2 and R3) are shown at the top of figure. R3P depicts the location of the probe used in Southern hybridisation. The location of NdeI sites and kanamycin resistance gene (unfilled arrow) are also shown. Genes involved in mobilisation, integration into recipient chromosomes, and those involved in conferring resistance to arsenate and cadmium are shown.

Figure 2

ICE Sde 3396 is transferable from SDSE, GAS and GBS. Arrows represent direction of transfer of the ICE into recipient strains. All three species are capable of acting as donors and recipients of the ICE. Transconjugants were initially identified on the basis of double antibiotic resistance phenotype. Transfer of the full ICE was confirmed by PCR amplification of DNA from Regions 1, 2 and 3 (data not shown).

Figure 3

ICE Sde 3396:km is chromosomally integrated in SDSE, GBS and GAS. (A) Southern hydridisation of NdeI restricted streptococcal chromosomal DNA probed with R3P. The presence of reactive bands greater than 8.0 kb in GAS and SDSE transconjugants is indicative of chromosomal integration of the ICE. (B) PCR amplification of the terminal region of ICESde3396:km and chromosomally encoded rplL gene from ICE-negative wild-type GBS (wt), and corresponding ICE-positive transconjugants (tc) from group B streptococcus, demonstrating chromosomal integration of the ICE in transconjugants.